Transiently restricting individual amino acids protects Drosophila melanogaster against multiple stressors

Nutrition and resilience are linked, though it is not yet clear how diet confers stress resistance or the breadth of stressors that it can protect against. We have previously shown that transiently restricting an essential amino acid can protect Drosophila melanogaster against nicotine poisoning. Here, we sought to characterize the nature of this dietary-mediated protection and determine whether it was sex, amino acid and/or nicotine specific. When we compared between sexes, we found that isoleucine deprivation increases female, but not male, nicotine resistance. Surprisingly, we found that this protection afforded to females was not replicated by dietary protein restriction and was instead specific to individual amino acid restriction. To understand whether these beneficial effects of diet were specific to nicotine or were generalizable across stressors, we pre-treated flies with amino acid restriction diets and exposed them to other types of stress. We found that some of the diets that protected against nicotine also protected against oxidative and starvation stress, and improved survival following cold shock. Interestingly, we found that a diet lacking isoleucine was the only diet to protect against all these stressors. These data point to isoleucine as a critical determinant of robustness in the face of environmental challenges.


Introduction
Nutrition is closely linked to health, and the more we explore this connection, the closer we come to being able to deliberately manipulate components of the diet to influence health and fitness.Nutrient restriction, in particular protein restriction, has been linked to increased lifespan, improved metabolic health and stress resistance in many model systems, usually at the cost of reproductive output [1][2][3][4][5][6][7].These effects can also be mimicked by restricting dietary amino acids [8][9][10][11][12].For instance, transient isoleucine deprivation enhances nicotine resistance and extends lifespan in Drosophila [13], methionine restriction enhances resilience to chemical and thermal stress in yeast, mice and human cells [14][15][16], and depriving mice of tryptophan protects them against ischaemic reperfusion injury [17].These beneficial effects of diet, particularly these short-term restrictions, have attracted interest for their potential to enhance human health.Current data indicate that evolutionarily conserved mechanisms mediate these effects [13,17,18], though little is known about the breadth of stress resistance these dietary manipulations afford and/or if there are costs beyond reproductive arrest that are associated with their implementation.These considerations, as well as understanding their mechanisms, are important when seeking to apply treatment protocols across species.
It is important to consider that seemingly beneficial dietary strategies may impose unintended costs, or trade-offs.One trade-off that is well described in the literature is between lifespan and reproduction; while lifespan is longest on low-protein, high-carbohydrate diets, reproduction in females is highest in intermediate-protein, intermediate-carbohydrate diets [19][20][21][22].Because these traits are optimized on different diets, this makes it difficult for animals to both live for a long time and have maximal reproduction.Using a similar logic, if short-term amino acid restriction can protect flies against nicotine, and prolong life, does this mean that it will protect them against other stressors too, or do these benefits trade off against other dimensions of stress resistance?
There are different types of biotic and abiotic stressors against which organisms must evolve strategies to resist, tolerate and/or avoid.In nature, flies and other animals frequently encounter stressors such as naturally occurring insecticides, temperature fluctuations and infections [23].It is also likely that an animal will encounter more than one stressor at a time.For example, winter is not only cold but also dry, so insects must tolerate both simultaneously to survive [24].It is therefore important that organisms launch multiple resistance phenotypes, even when sensing only one stressor, so they have the best chance to survive current and anticipated environments.
In this article, we present our findings on the way Drosophila melanogaster initiates a broad range of stress responses when experiencing short-term amino acid deprivation.Our study contributes to a deeper understanding of the complex interactions between nutrition and stress resistance, offering insights that could inform the development of personalized dietary strategies for promoting health and longevity.

Fly husbandry
Experiments were conducted using white-eyed D. melanogaster (strain Dahomey; wDah).Outbred wDah stocks are maintained in high-density population cages with continuous overlapping generations on a sugar yeast (SY) diet [25] (electronic supplementary material, table S1) at 25°C, ambient humidity and a 12 : 12 h light : dark cycle.Experimental flies were reared from egg to adult at a density of approximately 250-300 flies per bottle with 70 ml SY medium and mating status was standardized by keeping newly emerged adult flies in mixed cohorts on fresh SY diet for 2 days following eclosion (figure 1).Unless specified, experimental flies were mated, female, wDah.Two days after eclosing, flies were lightly anaesthetized with CO 2 and sorted by sex into vials containing a complete synthetic diet [26] (electronic supplementary material, table S2) in cohorts of 5 flies per vial.Flies were transferred to fresh food every Monday, Wednesday and Friday, unless otherwise specified.Experiments and stocks were maintained at 25°C, 60% humidity and a 12 : 12 h light : dark cycle.

Synthetic media preparation
Synthetic media were prepared as described by Piper et al. [26] containing the nutrients listed in electronic supplementary material, table S2.The complete synthetic diet contained an exome-matched ratio of amino acids [27] (FLYaa), and the other pre-treatment diets were prepared in the same way except with a reduced amount of the focal amino acid.Media were prepared in advance and stored for up to 4 weeks at 4°C.

Laced-media preparation
Survival under nicotine or paraquat exposure was measured using fly food that was laced with the respective drug.The nicotine-laced medium was prepared by aliquoting 100 µl of diluted free base nicotine (electronic supplementary material, table S3) into a vial containing 3 ml of cooled, gelled complete synthetic medium (final concentration of 0.83 mg ml −1 nicotine in vials).Paraquat-laced vials were similarly prepared by aliquoting 100 µl of diluted methyl viologen dichloride hydrate (electronic supplementary material, table S3) onto 2 ml of cooled, complete synthetic medium (final concentration of 10 mM paraquat in vials).Laced vials were then kept in a fabric cover at room temperature for 24-48 h to ensure an even dispersion of toxin throughout the food.Laced food was prepared only in sufficient volumes to match what was needed for immediate use, and only 24-48 h in advance of use, as these drugs lose potency over time.

Egg counting
When measuring fecundity, flies were first allowed to lay eggs in fresh vials for 24 h.Following this, flies were transferred to new vials and the vials containing eggs were photographed using a stereo microscope (Zeiss; Stemi 508) with an attached camera (Zeiss; Axioxam ERc 5 s).The photographs were then piped through an application that was made in-house by Jing J. Tan to count the number of eggs in each vial.This application is available for public use (see https://github.com/Eyehavelived/egg_counter).

Nicotine and paraquat exposure protocol
Following pre-treatment (figure 1), cohorts of 5 flies per vial were transferred into vials containing either 0.83 mg ml −1 nicotine or 10 mM paraquat.After 48 h in these toxin-laced vials, flies were transferred to freshly prepared toxin-laced vials.Once initially exposed to their respective toxin, survival was recorded at 7.00, 13.00 and 19.00 hours for 3 days, and then at 8.00 and 17.00 hours for 2 days, following which any remaining surviving flies were censored.Fly survival was recorded using the software DLife [28].

Starvation protocol
After they were pre-treated with amino acid deprivation (figure 1), flies were mildly anaesthetized with CO 2 (3 l min −1 for <5 min) and placed individually into wells of a 96-well tissue culture plate (Falcon; FAL353072) that contained 700 µl of 2% agar (Sigma Aldrich; A7002).Before the flies awoke, the lid was placed on the plate to contain single flies to individual wells.We fitted a custom-built plate scanning robot with a digital camera (Dino-Lite) and used this robot to photograph each well of each plate every hour.We sorted the images by well location and used the photos in series to determine when the fly stopped moving, at which point they were scored as dead.

Heat knockdown protocol
Following pre-treatment with their respective diets (figure 1) flies were individually transferred to 5 ml glass vials using a mouth-controlled aspirator.The vials were then submerged into a 39°C recirculating water bath that was heated by a digital thermos-regulator (Ratek; model TH5).Heat knockdown time was measured as the time taken, to the nearest second, until a fly was immobile.

Cold shock protocol
Flies were pre-treated with their respective diets (figure 1) and then individually transferred to 1.5 ml Eppendorf tubes using a mouth-controlled aspirator.The Eppendorf tubes were then submerged in a tank containing 50% propylene glycol (v/v, with water) that had been cooled to 0°C using a Thermoline liquid cooler (TRC-500).After 5 h at 0°C, flies were then transferred to 25°C to recover.Recovery time was measured as the time taken (to the nearest second) for flies to begin moving again.A small number of flies (11 out of 300) did not recover within 2 h, and they were considered dead and omitted from the analysis.All flies that recovered were transferred individually into vials containing a complete synthetic diet, and survival was recorded 2-3 times a day for 5 days using the software DLife [28].

Infection protocol
Wild-caught Enterococcus faecalis stocks [29] were stored at −80°C in Luria Bertani (LB) broth with 15% glycerol.To prepare bacteria to infect flies, a stock was first streaked onto an LB plate and grown overnight at 37°C.A single bacterial colony was then picked from this plate and grown overnight in 2 ml of LB broth, in an orbital shaker kept at 37°C rotating 200 times per minute.The overnight cultures were diluted with sterile phosphate-buffered saline (PBS, Ph = 7.4) to an optical density (OD A600 ) of 0.1.Pre-treated flies (figure 1), were lightly anaesthetized with CO 2 (3 l min −1 for less than 5 min) and injected with 0.1 OD A600 E. faecalis using the septic pinprick method [30].Control flies were instead injected with sterile PBS.Following infection, flies were transferred in cohorts of 5 to a complete synthetic diet, and survival was recorded 2-3 times a day using the software DLife [28].Infected and control flies were transferred to fresh food every day, and the old vials were photographed to measure daily fecundity.

Statistical analysis
All analyses were completed using R (v. 4.2.2) [31] and R Studio (v.1.4.1106)[32], and we created all plots using ggplot2 [33].All data and scripts are publicly available (see 'Data accessibility' below).General methods for experiments.Newly emerged adult flies were transferred to fresh sugar yeast (SY) food to mate for 2 days, then sorted into vials containing a complete synthetic medium at a density of 5 flies (of the same sex) per vial.Unless otherwise specified, flies were transferred to their pre-treatment diet on day 7 of adulthood.If fecundity was measured before exposure to the stressor, flies were transferred to fresh vials on day 13 of adulthood and the eggs in those vials were counted on day 14.
To determine whether the independent variables of a model could explain variation in the data, we initially analysed the models using a type II or III ANOVA from the package car [34].
Cox proportional-hazards modelling was used to analyse survival.To do this, we used the 'coxph' function from the package survival [35].
Differences in fecundity were analysed using a linear model (base R, 'lm') and post hoc comparisons from the emmeans [36] and multcomp [37] packages.
Linear models (base R, 'lm') were also used to model both the duration of pre-treatment and the dose of amino acid as a function of survival.To determine whether pre-treatment duration or availability of the focal amino acid significantly impacted survival, we analysed the model using the 'ANOVA' function from the package car [34].

Results
3.1.Pre-treatment diets that protect female flies from nicotine do not protect males In our previous work, we found that some diets lacking an essential amino acid protected female flies from subsequent nicotine poisoning [13].While female flies stop laying eggs when they are fed food without one or more essential amino acids, this protective effect cannot be attributed solely to a simple trade-off against reproduction.This is because neither a leucine or methionine dropout nor a diet missing all amino acids was protective, yet they still reduced egg laying to the same level as protective diets [13].As protection by diet cannot be simply explained by a trade-off against reproduction, we hypothesized that males-who are thought to expend less energy for reproduction [38]-could be afforded the same protective benefits of diets that increased female flies' nicotine resistance.To explore this, we pre-treated both sexes with a diet lacking isoleucine for 7 days, which we previously found to be the most protective pre-treatment regimen [13].We also pre-treated flies with a nutritionally complete diet to normalize results between males and females, and a protein-free diet to control for removing all amino acids.
When comparing males and females that were not pre-treated (complete diet), males had a greater nicotine resistance than fully fed females (figure 2a; p = 0.04).However, pre-treating flies with an isoleucine dropout diet for 7 days increased female, but not male, nicotine resistance (figure 2b; table 1).Removing all amino acids was equally detrimental to both sexes (table 1).These results indicate that there is sexual dimorphism in the stress resistance afforded by diet.

Restricting individual amino acids, but not all amino acids proportionately, is protective against nicotine exposure
Restricting protein in the diet of model organisms has repeatedly increased the consumer's resilience to stress [1].We were, therefore, interested in whether protein restriction could protect female flies against nicotine in the same way that single amino acid deprivation does.In all prior experiments, we have found that completely depriving flies of protein for short durations has not been beneficial.However, we have also found that the strength of the protective response varies with the identity of the amino acid, the degree of restriction and the duration of pre-treatment [13].It is therefore possible that manipulating total protein could be protective when it is restricted in a way that we have not yet explored.We hypothesized that feeding flies less protein, or starving them of protein for a shorter period of time, could protect them against nicotine in the same way that restricting a single amino acid does.
To do this, we pre-treated two separate cohorts of flies.The first cohort was fed one of seven diets in which all amino acids were restricted to 75, 50, 25 or 0% of the amount in the complete diet for 7 days prior to exposure to nicotine.The second cohort was fed a diet lacking amino acids/protein for shorter lengths of time before nicotine exposure.We found that both methods of protein restriction significantly impacted fecundity, which we measured in the 24 h prior to nicotine exposure (figure 1).We found that for every 6.66% that protein was reduced in the diet, egg laying was reduced by approximately 1 egg per female in the 24 h measured (F 4 = 81.49,p < 0.001).Similarly, consuming less protein by spending more time on a diet without protein also reduced fecundity by approximately 2 eggs per female over 24 h for every additional day without protein (F 4 = 76.79,p < 0.001).These decreases in fecundity are similar to what is observed when flies are deprived of a single essential amino acid [39].When we exposed these cohorts of flies to nicotine after pre-treatment, we found that, contrary to expectations, no level of protein restriction increased nicotine resistance, and reducing protein to 25 or 0% was detrimental (figure 3c; table 2; electronic supplementary material, figure S1a).Similarly, reducing the time that flies were starved of protein did not increase their nicotine resistance, and, as we previously found, starving them of protein for 7 days was detrimental (figure 3d; table 2; electronic supplementary material, figure S1b).These results suggest that the benefits of individual amino acid restrictions prior to nicotine poisoning are specific to individual amino acids and perhaps require the presence of one or more of the remaining 19 amino acids.

Assessing how availability and duration of methionine and leucine dilution impact nicotine resistance
Given that restricting all amino acids proportionately did not protect flies from subsequent nicotine poisoning, we decided to return to exploring the benefits of restricting individual amino acids.In particular, we were interested in discovering the conditions that conferred the greatest benefit when restricting different amino acids.We previously found that the conditions that provided the maximum protection when isoleucine was altered were different to the conditions when threonine is altered [13].Specifically, removing isoleucine from the diet for 7 days was the most protective, whereas threonine only needed to be reduced to 25% to show the greatest protection against nicotine.In our initial screen, where we removed each amino acid individually, we found that there was no effect of depriving flies of either methionine or leucine, whereas removing any one of the other 8 essential amino acids for 7 days provided some degree of protection.This was particularly curious because other research describes the benefits of restricting these amino acids.Methionine restriction is strongly associated with longevity and increased metabolic health in flies, worms and mice [40] and leucine is one of the three branched chain amino acids, and the other two, isoleucine and valine, both protected flies against nicotine when removed from the diet [13].As we have already shown that the intensity of restriction and the length of pre-treatment can impact the benefits of single amino acid restriction, we wanted to know whether there were conditions where flies were protected when we modified the availability of either methionine or leucine.
We individually restricted methionine or leucine to 75, 50, 25 or 0% of the amount in the complete diet.As we restricted the amount of these amino acids, we simultaneously modified the duration of dietary pre-treatment to 7, 5, 3 or 1 day prior to      nicotine exposure, resulting in a combination of 16 pre-treatment conditions plus a complete diet control.In the 24 h immediately before nicotine exposure, we measured the fecundity of these flies to further understand the relationship between toxin resistance and reproductive output.When we modified methionine, we found that the only condition that significantly altered egg laying was a diet completely lacking methionine for 7 days (figure 4a; table 3).Although methionine is an essential amino acid, this result was unsurprising because egg laying has previously been shown to exhibit a slower decline after methionine removal than that of the other amino acids [39].When we removed leucine from the diet, we observed a proportional reduction in egg laying, where fecundity was reduced by approximately 2 eggs per female in the 24 h measured for every additional day that flies spent on a leucine dropout (figure 4b; electronic supplementary material, table S4).Interestingly, we saw that a 25% leucine diet had a less steep decline in egg laying of approximately 1 egg per female per 24 h for every additional day spent on this diet and this decline appeared to level out after 5 days spent on the diet.Neither 50% (p = 0.14) nor 75% (p = 1) had a noticeable change in egg laying over time.Together, these data indicate that the flies were experiencing restriction for each of these amino acids to differing extents, and reinforce that they are likely to have internal reserves of amino acids on which they can draw to sustain reproduction [41].
When pre-treated flies were exposed to nicotine, there was an interaction (second-order polynomial) between the amount of methionine in the diet and how long the diet was fed to the flies that modified their survival (table 4; figure 4c).The flies that lived the longest when exposed to nicotine were fed a diet of 25% methionine for 7 days before they were poisoned (mean survival of 60.7 h on nicotine) which was protective when compared with the complete medium (mean survival of 46.1 h; p = 0.012).This result further uncouples nicotine resistance and reproductive capacity, as the fecundity of these flies did not differ from that of the fully fed controls.When leucine was modified in the diet, the duration of pre-treatment did not significantly impact survival, only the amount of leucine (table 4, figure 4d).The flies that survived the longest were fed a 50% leucine diet for 5 days (mean survival 57 h on nicotine), but this was not protective when compared with the complete diet (mean survival of 46.1 h; p = 0.09).These results again signal that fly physiology responds in a specific way to each amino acid and the degree to which it varies, and that there is not a simple trade-off between reproduction and survival under toxic conditions.

Diets change how flies respond to different forms of stress
Previously, we have explored how nicotine resistance can be modified by diet, though there are many ways to stress a fly, some of which have been linked to nutrition [42][43][44][45].Given that there is not necessarily an obvious relationship between diet and nicotine resistance, we wondered whether the diets that protect against nicotine could protect against a broad spectrum of stressors.To explore this, we selected a panel of diets that were the most protective for a focal amino acid against nicotine and used them as pre-treatment before exposure to various stressors.This panel included a 7-day treatment with either an isoleucine dropout, a 25% threonine diet or a 25% methionine diet and, although it was not protective against nicotine, we also included a 5-day, 50% leucine pre-treatment condition.As controls, we included a complete diet as a reference and a diet lacking all amino acids and only investigated the effects of these diets using female flies due to the differences we found between male and female responses to diet.

Oxidative stress (paraquat)
Paraquat (N,N′-dimethyl−4,4′-bipyridinium dichloride) is commonly used to induce oxidative stress in Drosophila.Paraquat reacts in vivo to ultimately produce a superoxide anion, which is a reactive oxygen species (ROS) that can cause damage to lipids, proteins and DNA [46].ROS can also be produced endogenously as a result of normal mitochondrial function [47], meaning that it is important for organisms to have systems to mitigate the effects of ROS.To see if we could potentially prime these systems with diet, flies were pre-treated with our panel of diets and then chronically exposed to 10 mM paraquat in their food.We found that diet-modified paraquat resistance (χ 2 5 = 60.0,p < 0.001), but only the isoleucine dropout pre-treatment protected flies against paraquat (p < 0.001; figure 5a; table 5).We also found that a diet lacking all amino acids reduced flies' capacity to resolve oxidative stress (p = 0.003).These data hint that there might be effects of isoleucine deprivation that are not elicited by other types of amino acid restriction.

Starvation
A well-established method for increasing starvation resistance in flies is nutrient restriction.This could be in the form of dietary restriction [48], protein restriction [49] or even single amino acid deprivation [50].This is hypothesized to result from flies responding to these diets by reducing their reproductive output and in turn, storing nutrients such as fats which can be used to maintain life when starved [43].When we starved flies after our single amino acid pre-treatments, we found that the diet affected survival (χ 2 5 = 74.9,p < 0.001).Specifically, we found that an isoleucine dropout diet, a 25% threonine diet or a 25% methionine diet increased starvation resistance (figure 5b; table 5).Flies that were pre-treated with either a protein-free diet or a 50% leucine diet responded to starvation no differently than the fully fed control flies.Given that we have shown that a 25% methionine diet does not reduce fecundity and increase starvation resistance and that a diet lacking amino acids reduces fecundity but does not protect against starvation, our data suggest that fecundity is not simply traded for starvation resistance.

Cold shock
In nature, organisms are faced with thermal challenges such as extremely cold or hot conditions to which they must evolve resistance or tolerance.Time to wake up after a chill coma is a trait that is often measured in the context of population plasticity in the face of climate change [51], but subsequent survival in the following days is not always measured.Here, we measured both.We found that the pre-treatment diet impacted both recovery time (χ 2 5 = 28.8,p < 0.001) and survival following cold shock (χ 2 5 = 53.2,p < 0.001).However, none of our pre-treatment diets improved chill-coma recovery time (figure 5c; table 5), in fact, a 25% methionine or a 50% leucine diet increased the time it took for flies to wake.However, we found that flies pre-treated with an isoleucine dropout, a 25% threonine or a protein-free diet lived longer in the 5 days following cold shock (figure 5d; table 5).When we examined the data across all pre-treatment groups, we discovered that there is an overall correlation between recovery time and survival post cold shock (p < 0.001, Pearson's correlation coefficient = −0.24).However, the pre-treatment diets that improved survival following cold shock did not differ in their recovery time from the complete diet.These results highlight that flies can recover at the same rate from chill coma, but this is not necessarily an indication of their health status, as many die in the following days.They also indicate that pre-treatment diets can improve the health status of flies following cold shock without influencing recovery time.

Heat knockdown
On the other end of the thermal spectrum from cold shock is heat shock.In the face of climate change, animals are facing increasing temperatures and so understanding their upper thermal limit is an important indicator of stress resistance with implications for population persistence [52].It is more typical, however, to manipulate the diet of flies during their development than during adulthood, and there is a link between larval diet and upper thermal tolerance [53,54].We were, therefore, interested to know whether there is a similar link between heat tolerance and our adult pre-treatment diets.We found that the pre-treatment diet affected heat knockdown time (χ 2 5 = 35.3,p < 0.001), though none of our pre-treatment diets enhanced heat tolerance when compared with the complete diet (figure 5e; table 5).In fact, flies that were pre-treated with a 25% threonine diet or a 50% leucine diet were more susceptible to heat knockdown than the complete diet.These results indicate that the mechanisms by which these diets protect flies against nicotine are not generalizable across all types of stress.

Infection with E. faecalis
We were also interested to know whether our diets could protect flies against a biotic stress.To do this, we opted to prick flies with live Enterococcus faecalis, a bacterium that naturally colonizes the gastrointestinal tract of flies [55].Enterococci are a leading cause of nosocomial infections in humans [56] and E. faecalis is routinely used to infect Drosophila and study host-pathogen interactions [29,57,58].When we infected flies with E. faecalis, we found that the pre-treatment diet overall did not impact survival (χ 2 5 = 9.6, p < 0.09).Though when we compared survival between different pre-treatment diets and the complete diet, we found that a diet lacking amino acids increased susceptibility to the pathogen (figure 5f; table 5).When we infected the flies, we were also interested to know whether the pre-treatment diet could impact fecundity under infection, as we expected that the energy that would be expended on laying eggs would need to be diverted to resolve the infection.However, we did not see any evidence for this, and flies that were infected had indistinguishable egg laying dynamics to control flies for each pre-treatment (figure 6; table 6 ; electronic supplementary material, table S5).Together, these results show that the ways that diet can protect against nicotine poisoning are not the same for infection with E. faecalis.

Discussion
In this article, we showed that there is sexual dimorphism in the protection afforded by short-term individual amino acid deprivation and that the protection cannot be mimicked by manipulating the total protein in the diet.Moreover, we found further evidence that there are different optimal pre-treatments for each amino acid to increase the stress resistance of the consumer.Finally, we showed that different pre-treatment diets could protect flies against different stressors to different extents.This research furthers our understanding of the benefits that are conferred by amino acid-restricted diets and offers insights into the complex relationship between nutrition and stress resistance.
In our previous work, we found that diets lacking an essential amino acid protected female flies from subsequent nicotine poisoning.It is particularly common in studies that investigate diet using fruit flies to only experiment with females, as changes in diet lead to rapid, observable changes in fecundity [59].Several studies have also observed larger effect sizes of diet on females than males which is generally attributed to females eating more than males to sustain their reproductive output [21,60].Given this information, we assumed that males would also receive the benefits of an isoleucine dropout, but perhaps to a lesser degree than females.This was not the case however, and instead we found that short-term isoleucine deprivation reduced the nicotine resistance of males.Interestingly, fully fed males were more tolerant to nicotine than fully fed females, which is surprising given that young females typically have greater resistance to a range of stressors than the same age males [61].This could reflect a difference in the initial investment capabilities of the sexes, as the females in our experiments were mated and so were committed to a greater reproductive investment than the males.2).Survival after cold shock was improved when flies were pre-treated with a diet lacking all amino acids (p < 0.001), an isoleucine dropout diet (p < 0.001) or a 25% threonine diet (p = 0.004).(e) Pre-treatment diets that lack all amino acids (p < 0.001)-or contain only 25% threonine (p = 0.002) or 50% leucine (p = 0.002)-increase susceptibility to heat knockdown.(f) Flies that were pre-treated with a diet lacking amino acids were more susceptible to infection with E. faecalis.The number of individuals varied between 29 and 50 flies per pre-treatment group for each experiment (table 5).***p < 0.001, **p < .01,*p < 0.05.It is also possible that these differences could reflect the difference in food, and so toxin, consumption by male and female flies [62].Differentiating between these possibilities could be addressed by administering nicotine using capillary feeders that would permit precise quantification of toxin ingestion [63,64].It is also possible that the combinations that benefit females simply do not benefit males, and that the sexes have different responses to diet.Given that the identity of the focal amino acids, as well as the degree of restriction and pre-treatment duration all interact to impact female nicotine resistance, it is likely that sex also modifies this response.
To understand this, we should investigate the differences in molecular responses to isoleucine deprivation between males and females.Previous research has shown sex-specific transcriptional responses across high-and low-protein diets, implying that the output of nutrient-sensing pathways is different between male and female flies [65].Male and female flies also differ in basal activity levels of processes such as autophagy in the intestinal enterocytes, where male flies have relatively high levels of autophagy compared with females [66].This explains the sexual dimorphic effects on lifespan extension from rapamycin treatment in flies, as rapamycin extends lifespan in female flies by elevating the levels of autophagy in female gut enterocytes to reduce age-related intestinal dysplasia.Conversely, males suffer a lower incidence of age-related intestinal dysplasia, apparently due to higher basal levels of autophagy in gut enterocytes, and therefore do not experience the same benefits to lifespan from rapamycin treatment as females [66].Future studies on the sexually dimorphic effects of diet and stress tolerance should not only compare the molecular responses between males and females following dietary treatment but also consider that basal activity levels of cellular processes might result in sexually dimorphic effects.It is important that we understand the molecular mechanisms driving differences in physiological responses to diet between sexes if we are to make any suggestions about diet for improving human health in the future.
The benefits of chronic protein restriction, including both lifespan and health span extension, have been documented across a wide range of taxa [1,67].Recently, the acute benefits of protein restriction have also come to light.For example, mice are more  resistant to hepatic and renal ischaemic reperfusion injury, which are models of liver and kidney surgery, respectively, when they have been fed protein-restricted pre-treatment diets [5,18].Similarly, protein restriction protects flies against hydrogen peroxide, an oxidizing agent, and also protects old flies against infection with E. faecalis [2].Since restricting an individual amino acid protects flies against nicotine, it was surprising to find that protein restriction by restricting all dietary amino acids did not also protect flies.It is possible, therefore, that flies require one or more of the remaining non-focal amino acids to be supplied in the diet to establish resistance.A potential contender for this amino acid is leucine, since no methods of leucine restriction protected flies against nicotine.It would be interesting to know if restricting all amino acids except leucine is beneficial, or if the benefits of combining nicotine-protective diets (i.e.food lacking isoleucine, with 25% threonine, 25% methionine and containing leucine) is more protective than any single amino acid restriction alone.The current framework of research mainly focuses on observing the protective effects of protein or dietary restriction [68,69], but our work emphasizes that dietary restriction and individual amino acid restriction are not equivalent, and sometimes amino acid restriction is protective when other types of restriction are not.Thus, there is merit in altering the levels of individual dietary amino acids even when there are no, or negative, effects of other dietary restrictions.
A common theme from our data is that short-term isoleucine deprivation was the most common way to protect flies against stressors.Isoleucine restriction has recently garnered more attention than that of other amino acids, because of the generally protective effects that result from restricting its intake.When it is restricted in the diet, female flies live longer [70] and so do male and female heterogenous mice, who also have improved metabolic health [71].Metabolic health can also be restored in obese mice by restricting isoleucine intake [72].We have also recently found that female flies subjected to two short bouts of isoleucine deprivation have extended lifespan [73].A potential explanation for this is that isoleucine deprivation triggers protective systems that bolster defence against a broad spectrum of damage threats and that these mechanisms overlap with those that improve lifespan.One possibility is a link between detoxification capacity and longevity; detoxification genes are strongly upregulated in long-lived insulin mutants [74] and insulin mutant flies are more resistant to dichlorodiphenyltrichloroethane (DDT) [75].Since flies were not protected by removing isoleucine at the same time as the other amino acids (i.e.no protein treatment), we propose that protection requires new protein synthesis.New proteins would surely require isoleucine and this could possibly be made available by recycling amino acid stores via protein breakdown [41].If this is the case, then these stored amino acids must somehow be reserved for somatic protection, rather than for use in egg production, which ceases when any essential amino acid is removed from the diet [39,59].Studies tracking the fate of labelled amino acids into protein during isoleucine deprivation could be revealing, both for understanding the systems that are triggered to enhance stress resistance and for identifying protein synthesis that is required to sustain lifespan.
Another factor that animals encounter in the wild is changes in temperature.It is particularly important for ectotherms to be aware of cues that signify a temperature change since they cannot regulate their own body temperature, and so must respond physiologically to ensure survival [76].Interestingly, we found nicotine-protective diets that reduced fecundity also increased survival following cold shock.This trade-off could be explained by the availability of sterols, essential micronutrients that modulate cell membrane fluidity [77], which are speculated to be important in mitigating mechanical injury to cell membranes during cold shock [78].Flies that are fed more cholesterol during development can have enhanced cold tolerance [79,80] and the trade-off between lifespan and reproduction can be rescued by supplementing cholesterol [81], allowing flies to have long lives and high fecundity.Taken together, it is possible that reduced fecundity due to essential amino acid deprivation increases body sterol stores, and this modifies cell membranes in a way that protects flies against acute cold shock.If this were the case, we would expect that supplementing adult flies with cholesterol would also protect them against cold shock, even when fed a high-protein diet.
We also found that there was some overlap between diets that protected against survival following a cold shock and starvation.This could be explained by the changing of seasons.Winter is associated with reduced temperatures and availability of nutrition, and evolving resistance to both simultaneously should be adaptive.Interestingly, flies that are evolved for chill coma recovery have higher levels of phosphatidic acids, but not triacylglyceride (TAG) or lipid levels [82].We found that the TAG levels of flies that were pre-treated with an isoleucine dropout or a 25% threonine diet were not different from controls fed a complete diet, but both of these pre-treatment diets protect against starvation and cold stress.It would be interesting to look at the levels of phosphatidic acids in our pre-treated flies as a possible mechanism for cross-protection against starvation and cold stress.
When selecting the stressors to expose flies to, we wondered whether there might be diets that increased resistance to one stress while trading-off resistance against another.This would assume that the response to a diet that increases resistance to one stress somehow competes with the demands of mitigating a different stress.However, we only investigated how dietary pre-treatments that offer protection against nicotine affect responses to other stressors, and we did not see a consistent trade-off in these experiments.This shows that the mechanisms by which diet can protect against nicotine poisoning are not consistently antagonistic to the mechanisms that protect against the other types of stress that we measured.
Previously, we have found that rapamycin, a drug that inhibits the target of rapamycin (TOR), protected fully fed flies against nicotine to the same degree as isoleucine deprivation [13].As TOR is a master regulator of growth, which detects cellular levels of amino acids and is inactivated by low levels of amino acids [83], we assumed that both isoleucine deprivation and rapamycin achieved nicotine resistance through TOR suppression.However, other studies have shown that rapamycin treatment improves the heat tolerance of wild-type flies [84] and can improve the maximum thermal temperature (CTmax) withstood by Drosophila Genetic Reference Panel (DGRP) flies [85].We therefore anticipated that short-term isoleucine deprivation would also increase heat tolerance in our flies, but it did not.Interestingly, other work has also shown that flies under dietary protein restriction are less heat tolerant [68].While dietary protein restriction and rapamycin treatment are thought to increase lifespan through TOR suppression [86,87], these data suggest that the molecular changes induced by rapamycin treatment are overlapping with those induced by isoleucine or protein restriction, but not the same.This idea is supported by recent research, where rapamycin treatment or restricting a single amino acid has vastly different effects on the hepatic metabolome, epigenome and transcriptome of mice [88].Interestingly, this study also highlights isoleucine deprivation as being distinctly different from either leucine or methionine deprivation, which both had similar effects on the metabolome and transcriptome.By combining phenotypic data with -omics-style approaches, we may be able to decipher the different responses to dietary and pharmacological interventions and understand how they can achieve beneficial physiological outcomes, such as resistance to various stressors and enhanced lifespan.This could ultimately lead to tailored dietary interventions that have precise quantities of each nutrient to best regulate molecular and metabolic pathways to achieve the desired outcome.

Conclusion
Our study sheds light on the intricate relationship and trade-offs between nutrition and stress resistance in D. melanogaster.We have demonstrated that short-term amino acid restrictions, particularly isoleucine deprivation, can protect against various stressors, including nicotine poisoning, oxidative stress, starvation and cold shock.We observed sexual dimorphism in the response to dietary manipulation, with females, but not males, benefiting from isoleucine deprivation.Our findings also highlight the differences between total protein restriction and individual amino acid restriction, emphasizing the importance of considering specific amino acids in diet manipulation studies.Future investigations into the molecular responses to dietary interventions and their implications for stress resistance across multiple stressors will provide valuable insights into optimizing health span and resilience across species.

Figure 1 .
Figure 1.General methods for experiments.Newly emerged adult flies were transferred to fresh sugar yeast (SY) food to mate for 2 days, then sorted into vials containing a complete synthetic medium at a density of 5 flies (of the same sex) per vial.Unless otherwise specified, flies were transferred to their pre-treatment diet on day 7 of adulthood.If fecundity was measured before exposure to the stressor, flies were transferred to fresh vials on day 13 of adulthood and the eggs in those vials were counted on day 14.

Figure 2 .
Figure2.Pre-treatment with a diet lacking isoleucine protects female, but not male, flies against nicotine poisoning.Male and female flies were pre-treated with one of three diets-isoleucine dropout, no amino acids (AAs) or a complete diet-before chronic exposure to 0.83 mg ml −1 nicotine.(a) Survival curves of flies immediately after introduction of nicotine.(b) Survival time of pre-treated flies that has been normalized to survival of flies fed a nutritionally complete synthetic diet (red horizontal line ± s.e.indicated by dashed red lines), circles represent mean lifespan for each condition ± s.e.In females, flies that were pre-treated with a diet lacking amino acids had reduced nicotine resistance (p < 0.001), whereas pre-treatment with an isoleucine dropout diet protected flies from nicotine (p = 0.02).Compared with the control diet, male flies were more susceptible to nicotine when they were pre-treated with either a diet lacking amino acids (p < 0.001) or isoleucine alone (p = 0.02).There were n = 49-50 flies per pre-treatment group for each sex.***p < 0.001, **p < 0.01, *p < 0.05.

Figure 3 .
Figure3.Restricting all amino acids does not protect flies against nicotine.Flies were either pre-treated with diets where all amino acids were reduced for 7 days, or where all amino acids were absent for 1, 3, 5 or 7 days before chronic exposure to 0.83 mg ml −1 nicotine.(a) Mean number of eggs (± s.e.) per fly in the 24 h before nicotine exposure when the concentration of amino acids is altered and (b) when all amino acids are removed for various days.(c) Survival time of pre-treated flies that has been normalized to the survival of flies fed 100% of amino acids (red horizontal line ± s.e.indicated by dashed red lines), circles represent mean lifespan for each condition ± s.e.Flies tolerated nicotine less when they were pre-treated with diets that had 25 or 0% of the amino acids found in the complete diet (p < 0.001).(d) Survival time of pre-treated flies that has been normalized to the survival of flies that were fed a complete diet (red horizontal line ± s.e.indicated by dashed red lines), small circles represent the mean lifespan for each replicate and large circles represent the group mean.Flies were less resistant to nicotine when they were fed a diet lacking all amino acids for 7 days before exposure.***p < 0.001, **p < 0.01, *p < 0.05.

Figure 4 .
Figure 4. Amino acid identity and availability modify protection against nicotine stress.Flies were pre-treated with diets containing one of five amounts of either methionine or leucine (0, 25, 50, 75 or 100%) for either 7, 5, 3 or 1 day before fecundity was measured and flies were chronically exposed to 0.83 mg ml −1 nicotine.(a) When dietary methionine was modified, only a diet lacking methionine for 7 days reduced fecundity (b) However, restricting dietary leucine resulted in females laying approximately two fewer eggs per 24 h for every day she spent on a leucine dropout (95% CI [−2.57,−1.76]),one fewer egg per 24 h for every day she spent on a 25% leucine diet (95% CI [-1.64,−0.84]),or no decline over time when fed 50% or more leucine (p > 0.1).(c) Survival, when methionine was modified, was influenced by both the amount of methionine and the duration of pre-treatment (table4), with a 25% methionine for 7 days offering the greatest protection (p = 0.012).(d) Leucine dose (p = 0.02) but not pre-treatment duration (p = 0.59) significantly impacted survival on nicotine, although the longest-lived flies-that were fed a 50% leucine diet for 5 days-were not protected (p = 0.09).For each combination of pre-treatment duration and available amino acid, n = 50 flies.Pre-treatment with 100% of each amino acid could not differ in duration time and n = 50 flies for this group in total.The survival data have been normalized and represented in electronic supplementary material, figureS2to show variation in survival.

Figure 5 .
Figure 5. Manipulating individual dietary amino acids can differentially change how flies respond to stress.Flies were pre-treated with one of six diets before being exposed to a physical stress.Survival is represented as the difference between pre-treated flies and the controls, which were fed a nutritionally complete diet (red horizontal line ± s.e.indicated by dashed red lines), circles represent mean lifespan for each condition ± s.e.(a) Flies that were pre-treated with an isoleucine dropout were more resistant to 10 mM paraquat (p < 0.001), and removing all amino acids reduced paraquat resistance (p = 0.003).(b) Starvation resistance was increased by pre-treating flies with an isoleucine dropout (p < 0.001) or a diet containing either 25% threonine (p < 0.001) or methionine (p < 0.05).(c)When cold-shocked flies were transferred to room temperature, the flies that were pre-treated with either a 25% methionine diet (p < 0.001) or a 50% leucine diet (p = 0.01) took longer to recover.(d) However, the survival of these flies following cold shock was no different from the complete diet (p > 0.2).Survival after cold shock was improved when flies were pre-treated with a diet lacking all amino acids (p < 0.001), an isoleucine dropout diet (p < 0.001) or a 25% threonine diet (p = 0.004).(e) Pre-treatment diets that lack all amino acids (p < 0.001)-or contain only 25% threonine (p = 0.002) or 50% leucine (p = 0.002)-increase susceptibility to heat knockdown.(f) Flies that were pre-treated with a diet lacking amino acids were more susceptible to infection with E. faecalis.The number of individuals varied between 29 and 50 flies per pre-treatment group for each experiment (table5).***p < 0.001, **p < .01,*p < 0.05.

Figure 6 .
Figure 6.Pre-treatment diet did not influence fecundity during infection.Flies were pre-treated with one of six diets before being infected with E. faecalis and transferred onto a complete diet.The trends in egg laying between infected (solid lines) and control (dashed lines) flies were not different from each other across the pre-treatment diets.The number of individuals for each condition was 30 flies.Each point (circle or triangle) represents the mean number of eggs laid per fly per replicate vial over the 24 h measured.

Table 1 .
Differences in survival between flies that were pre-treated with a diet lacking amino acids (no AAs) or an isoleucine dropout diet compared with flies that were fed a complete diet, separated by sex.Summary of Cox proportional-hazards modelling.Confidence level = 95%.royalsocietypublishing.org/journal/rsob Open Biol.14: 240093

Table 2 .
Differences in survival between flies that were pre-treated with diets that had reduced, or no protein compared with flies that were fed a nutritionally complete diet.Summary of Cox proportional-hazards modelling.Confidence level = 95%.

Table 3 .
ANOVA tables for the models that best reflect the relationships between egg production, duration of pre-treatment, focal amino acid and the level that amino acid was restricted to.Both models: eggs laid per fly ~ duration × available amino acid.

Table 4 .
ANOVA tables for the models that best reflect the relationships between nicotine resistance, duration of pre-treatment, focal amino acid and the level that amino acid was restricted to.Methionine model: age ~ poly(duration, 2) × poly(available methionine, 2).Leucine model: age ~ duration × available leucine.

Table 5 .
Summary table for survival of pre-treated flies compared with control flies across multiple stressors.Control flies were fed a nutritionally complete diet.Summary of Cox proportional-hazards modelling.Confidence level = 95%.

Table 6 .
ANOVA table for the model that looks for interactions between the number of eggs laid by infected and control females: eggs laid per fly ~ pre-treatment diet × infection status × time after infection.